Tobacco use causes five million deaths worldwide annually and is the leading cause of preventable mortality in the world. Nicotine is the addictive component of tobacco that binds to and activates a family of ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs). Activation of the receptors in the dopaminergic mesocorticolimbic reward pathway is thought to underlie the initiation of addiction whereas signaling through nAChRs in the habenulo-interpeduncular pathway that feeds into the reward pathway is thought to play a key role in eliciting nicotine withdrawal symptoms. One of the challenges in understanding nicotine's effect on nAChRs arises from the existence of multiple nAChR subtypes, each exhibiting unique electrophysiological properties and varying affinities for nicotine. Eleven mammalian neuronal nAChR subunits have been identified (?2-??7, ?9, ?10 and ?2-??4). Five subunits co-assemble to form receptors with the subunit composition of each channel determining its pharmacological and biophysical properties. Chronic nicotine exposure alters the expression of nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear, although transcriptional mechanisms are not thought to be involved. A growing body of evidence indicates that nicotine and cigarette smoke alters the expression of small 21-24 nucleotide long regulatory molecules, referred to as microRNAs (miRNAs). miRNAs are predicted to regulate the majority of all mammalian protein coding genes, typically by binding to complementary sites in the 3'- untranslated regions (3'-UTRs) of target mRNAs and guiding them to an RNA-induced silencing complex (RISC). To date, one miRNA, miR-1, has been reported to target nAChRs in C. elegans, leading to changes in nAChR function. Little is known regarding miRNA regulation in the context of mammalian nAChRs or nicotine addiction. We have preliminary data from a miRNA analysis using laser-captured material from mice chronically treated with nicotine that suggest: 1) a decrease in expression of two miRNAs, miR-7a and miR-9, in the ventral tegmental area (VTA) and 2) an increase in expression of the same two miRNAs in the medial habenula when compared to saline-treated animals. There was no change in their expression in the interpeduncular nucleus. Interestingly, potential targets of miR-7a and miR-9 include members of the nAChR family as well as other components important in the reward pathway. Our preliminary studies indicate that miR-7a regulates expression of the nAChR ?6 subunit via interactions with a single miR-7a recognition element in the 3'-UTR of the ?6 gene. These data form the basis of this application with the over-arching hypothesis that post-transcriptional regulation of nAChRs by miRNAs modulates nicotine-associated behaviors. The first Aim will test the hypothesis that miR-7a and/or miR-9 modulates nicotine reward-associated behaviors as well as nAChR expression and function. The second Aim will test the hypothesis that miR-7a and/or miR-9 modulates nicotine withdrawal-associated behaviors. The final Aim will test the hypothesis that miR-7a and/or miR-9 interacts with the 3'-UTRs of nAChR subunit genes in a functionally relevant manner. The results from these experiments will provide unique insight to the molecular underpinnings of nicotine addiction. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to understand the role of small regulatory RNA molecules in nicotine-mediated behaviors. Despite evidence indicating that expression of these molecules is altered by nicotine treatment, little is known regarding their function in this context. The proposed work will substantially close this gap in knowledge.